Published April 1, 1971
FINE STRUCTURE OF SMOOTH MUSCLE CELLS
GROWN IN TISSUE CULTURE
GORDON R . CAMPBELL, YASUO UEHARA, GERDA MARK,
GEOFFREY BURNSTOCK
and
From the Department of Zoology, University of Melbourne, Victoria, Australia
ABSTRACT
INTRODUCTION
Ultrastructural analysis of cultured striated muscle has played an important part in investigations
of the morphogenesis of myofilament formation
(1, 9, 10, 16) and the origin of the transverse
tubular or T-system (15, 22) .
Although smooth muscles have been grown in
tissue culture and studied with the light microscope since as early as 1911 (29), no reports on
their ultrastructure have been found . In the present study, both phase-contrast microscopy and
electron microscopy have been used to study
cultured smooth muscle cells of chicken gizzard .
Their fine structure is compared with that of adult
(2) and embryonic smooth muscle (3, 54) .
sociated into a cell suspension by using both collagenase and trypsin .l
In the present study, only 10- and 16-day old
embryos were used, but a wider survey is at present
under way .
The cells were grown on both the uncoated plastic
surface of Falcon cooper dishes (Falcon Plastics, Los
Angeles, Calif. ; supplied by Gateway International,
Los Angeles, Calif .) and on the collagen-coated (6)
glass surface of Sight Tissue Chambers, Sight Instruments, Hawaiian Gardens, Calif., a modification of
the original Rose chamber (41) . The cultures were
maintained with medium 199 (45) supplemented with
10% fetal calf serum and were examined in the living
state with phase-contrast optics and time-lapse microcinematography. 3-5-day cultures were fixed for
MATERIALS AND METHODS
The gizzards of 10-16-day old chick embryos were
stripped of the outer serosa and inner stratum compactum layer, and the muscular tissue was dis-
Mark, G ., J . Chamley, and J . McConnell . 1970 .
Vertebrate smooth muscle cells in tissue culture . In
preparation .
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The fine structure of smooth muscle cells of the embryo chicken gizzard cultured in monolayer was studied by phase-contrast optics and electron microscopy . The smooth muscle
cells were irregular in shape, but tended to be elongate . The nucleus usually contained
prominent nucleoli and was large in relation to the cell body . When fixed with glutaraldehyde, three different types of filaments were noted in the cytoplasm : thick (150-250 A in
diameter) and thin (30-80 A in diameter) myofilaments, many of which were arranged
in small bundles throughout the cytoplasm and which were usually associated with dark
bodies ; and filaments with a diameter of 80-110 A which were randomly orientated and
are not regarded as myofilaments. Some of the aggregated ribosomes were helically arranged . Mitochondria, Golgi apparatus, and dilated rough endoplasmic reticulum were
prominent . In contrast to in vivo muscle cells, micropinocytotic vesicles along the cell
membrane were rare and dense areas were usually confined to cell membrane infoldings .
These cells are compared to in vivo embryonic smooth muscle and adult muscle after treatment with estrogen . Monolayers of cultured smooth muscle will be of particular value in
relating ultrastructural features to functional observations on the same cells .
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FIGURES 1-4
Phase-contrast photographs of living cells . Scale 50
m, smooth muscle cells ;
FIGURE 1
f,
j.I
(see Fig . 4 a) applies to all figures .
fibroblasts . X 480 .
Muscle cell and fibroblasts . 1 day old culture of enzyme-separated cells from a 16 day embryo
chick gizzard .
FIGURE 2
Two muscle cells in end-to-end contact . 1 day old culture of enzyme-separated cells from a
17 day embryo chick gizzard .
FIGURE 3
Branched contracting muscle cells . 1 week old explant culture of cells from a 17 day embryo
chick gizzard .
FIGURE 4 a
Muscle cells from a 1 day old culture of enzyme-separated cells from a 10 day embryo
chick gizzard . These cells were actively contracting .
FIGURE 4 b
Muscle cells from same culture as Fig . 4 a after 5 days in culture . Note the ribbon shape
of the cells.
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FIGURE 5 Lower power electron micrograph of cultured smooth muscle cells from 10 day old embryonic
chicken gizzard 4 days in culture. Bundles of myofilaments (M) can be observed with associated dark
bodies (db) . The nucleus (N) has a smooth profile and contains a nucleolus (n) ; m, mitochondria ; G,
Golgi apparatus ; r, dilated endoplasmic reticulum with free ribosomes . X 20,000 .
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Published April 1, 1971
electron microscopy in collidine-buffered 2 .5% glutaraldehyde (pH 7 .4) for 15 min, washed in buffer
for 5 min, then postfixed in buffered 1 % osmium
tetroxide and in 2% uranyl acetate for 1 hr (49) .
After rapid dehydration through a graded series of
alcohols, the cultures were embedded in Araldite
(Ciba Products Co ., Summit, N .J .) . Infiltration
with a mixture of propylene oxide and Araldite was
not used, as propylene oxide dissolved the cooper
dishes and was found unnecessary with a cell monolayer . After 2 days at 60 °C the cultures were split
off the cover slips, and muscle cells selected before
fixation by phase-contrast microscopy were mounted
on rods (27) . Thin sections were cut on a HuxleyCambridge ultramicrotome, stained with lead citrate
(51) and examined in an Hitachi HU 11B electron
microscope .
shape, a large, pale nucleus, and several nucleoli
of variable size (Figs . 1-4) . These cells moved
about the culture and changed their shape freely
when seen in time-lapse cinematography . The
second type of cell was identified as smooth muscle by its occasional spontaneous contractility .
When taken from a 16 day embryo, these cells
were characteristically bipolar, 160-260 u long,
often forked at the ends (Fig . 3), with a small
oval nucleus, one to two small, regular, dense
nucleoli, and a more phase-dense appearance
than the fibroblasts .
Muscle cells from the 10 day gizzard were at
first irregular in shape and approximately 100 µ
long . After a few days in culture, they became
ribbon shaped and resembled more closely the
bipolar 16 day cells (Fig . 4, a and b) .
OBSERVATIONS
Electron Microscopy
Two main morphological cell types were clearly
distinguished after 1-2 days in culture . The first
resembled a classical fibroblast, with an irregular
Cells in cultured gizzard were identified as
smooth muscle by the presence of filaments with
associated dark bodies which were similar to
Longitudinal section through smooth muscle cell showing lipid droplets (L) and vesicles
in diameter (v) . M, myofilaments ; m, mitochondria ; r, endoplasmic reticulum ; arrows, microtubules. X 32,000 .
FIGURE 6
500-700 A
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Phase-Contrast Microscopy
Published April 1, 1971
cipitated . An accumulation of lipid droplets was
seen within some cells (Fig . 6) . Small vesicles
500-700 A in diameter and coated vesicles 500800 A in diameter were prominent throughout the
cytoplasm (Fig . 6) .
Electron-opaque particles about 200 A in
diameter were very abundant and were scattered
randomly throughout the cytoplasm (Fig . 7) .
These were of the same uniform size and appearance as ribonucleoprotein granules attached to
the membrane of the endoplasmic reticulum and
appeared to be free ribosomes . A number of
these ribosomes were in the form of polysomes .
Sometimes a number of ribosomes showed a
pattern suggesting a helical arrangement, where
the center-to-center distance between adjacent
ribosomes was about 220 A (Fig . 8) . These
polysomal helices appeared to have a random
orientation with respect to the myofilament
bundles, and were sometimes closely associated
with small filaments less than 30 A in diameter .
These filaments often extended into and inter-
FIGURE 7 Area containing an abundance of free ribosomes and polysomes . Some of these ribosomes are
in helical configurations (h) . r, endoplasmic reticulum ; M, myofilaments . X 48,000 .
CAMPBELL, REBARA, MARK, AND BURNSTOCK
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the myofilament-dark body arrangement seen
in both embryonic and adult smooth muscle cells .
However, in contrast to adult in vivo muscle,
where myofilaments extend throughout the cytoplasm, the myofilaments in the cultured cells
were confined to bundles in limited areas . Many
cells from the 10 day old cultured gizzard showed
small, randomly placed bundles (Fig . 5), whereas
in the 16-day old gizzard cells the bundles were
larger. The nuclei of both 10- and 16-day old
gizzard cells were slightly elongated with a smooth
profile and usually contained prominent nucleoli .
Mitochondria, granular endoplasmic reticulum,
and Golgi apparatus were particularly conspicuous and, unlike adult smooth muscle (2, 11),
appeared to be distributed throughout the whole
area of cytoplasm and were not restricted to the
cell margin or perinuclear region (Figs . 5-7) .
The granular endoplasmic reticulum was often
dilated with a cisternal diameter of up to 1 p .
The cisternae usually contained a moderately
electron-opaque material, sometimes finely pre-
Published April 1, 1971
mingled with the myofilament bundles . Particles
were often seen within the myofilament bundles
(Fig . 9) . The particles were slightly smaller and
less electron opaque than ribosomes, although
they may represent glancing sections through
ribosomes . However, it is possible that there is a
gradation in both electron opacity and size between particles within the myofilament bundles
and free ribosomes outside .
It has been suggested that both thick and thin
FIGURE 9 Longitudinal section of myofilament bundle showing ribosomal-like particles . Some profiles
are 250-300 A in diameter (circle), others are much smaller (double headed arrows) . db, dark body ; arrows,
100 A filaments . X 90,000 .
Dark bodies (db) in association with 100 A filaments (arrows) . The dark body appears to
consist of a dense matrix containing numerous, less electron-opaque subunits . M, myofilaments . X 130,000 .
FIGURE 10
FIGURE 11 Shows electron-opaque material
myofilaments . X 160,000.
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(arrow)
THE JOURNAL OF CELL BIOLOGY • VOLUME 49, 1971
in association with a membranous system . M,
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Helical arrangement of ribosomes . Small
filaments (arrows) are seen in close association with
these ribosomes. These extend into the myofilament
bundle (M) . t, microtuble . X 150,000 .
FIGURE 8
myofilaments are present in in vivo smooth
muscle (8, 25, 31, 46) . A few cultured cells contained both thick (150-250 A in diameter) and
thin (30-80 A in diameter) myofilaments, although the electron opacity and thickness of
these thick filaments were quite variable throughout their length (Fig . 16) . The majority of cells
contained only the thin filaments (Figs . 5, 9) .
Dark bodies were found closely associated with
the myofilament bundles (Fig . 5) . They appeared to be similar, although often smaller,
than those of adult tissue which are reported to
have a length of 700-2000 A (8) . They were
usually found within bundles of myofilaments,
except for a few on the periphery of bundles .
Under high resolution these dark bodies consisted
of a dense matrix containing numerous less
electron-opaque units about 70-100 A in diameter (Fig . 10) .
Another type of filament was found throughout
the cultured cells . These filaments were 80-110 A
in diameter and, unlike myofilaments, were more
uniformly electron opaque . Sometimes these filaments were in the form of clusters or networks
(Fig . 12) . These networks were randomly orientated and the filaments appeared to be similar
to the intermediate filament of cultured skeletal
muscle (termed "100 A filaments" [23]) . Some
of these networks contained electron-opaque
areas approximately 400 A in diameter and 700
A in length (Fig . 13) . These areas resembled the
dark bodies of the myofilament bundles . The
100 A filaments were also sometimes found in
association with dark bodies between discrete
bundles of myofilaments (Fig. 10) . Here, the
100 A filaments appeared to pass from within
the substructure of the dark body to the myofilament bundles .
Microtubules 250 A in diameter were scattered throughout the cytoplasm (Figs . 6, 8) .
The walls were smooth, 40 A thick, and uniform
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CAMPBELL, UEHARA, MARK, AND BIIRNSTOCK
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THE JOURNAL OF CELL BIOLOGY
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VOLUME 49, 1971
Published April 1, 1971
FIGURE 14
Area containing elaborate membranous network forming tubular and oblate profiles (T) .
Area containing elaborate membranous network forming tubular and oblate profiles (T),
part of which has become flattened to form cisternae (C) . A few ribosomal particles appear to be associated with the cytoplasmic side of a cisternal membrane (arrow) . X 36,000 .
FIGURE 15
in longitudinal section. They are similar to those
described in adult tissue (26) .
An unusual feature characteristic of some cultured smooth muscle cells, but not seen in in vivo
smooth muscle, were areas containing elaborate
membrane networks forming tubular (300-400
A in diameter) and oblate profiles (Figs . 12, 14,
15) . These networks were usually confined to the
periphery of the cell and showed some similarity
to differentiating T-system tubules of skeletal
muscle in tissue culture (15, 22), although in
smooth muscle they had a smaller diameter and
were not as well organized . Fig . 15 shows one
of these tubular systems, part of which has become
flattened to form cisternae . Some opposing membranes of these cisternae were closely applied to
form a specialized structure, while a few ribosomal particles appeared closely related to the
cytoplasmic leaflets of other cisternal membranes.
Micropinocytotic vesicles on the surface of
cultured smooth muscle cells were rare and generally did not alternate with dense areas or patches
as in the adult (8) . These dense areas were most
commonly found along distinctive infoldings of
the cell membrane (Fig . 16), and extended up to
800 A into the cell . Myofilaments ran alongside
the dense areas and often seemed to pass into their
matrix . Some electron-opaque material was sometimes seen in association with membranous systems throughout the cell (Fig . 11) .
Cultured smooth muscle cells in close contact
with each other were usually separated by a gap
of 200 A, increasing to 300-400 A when basement
membrane substance was present . This is somewhat less than the basement membrane-filled
Clusters or networks of filaments 80-110 A in diameter found within some cultured smooth
muscle cells . T, network of tubules ; arrows, part of tubular system . X 78,000.
FIGURE 12
Network of filaments containing electron-opaque areas approximately 400 A by 700 A
m, mitochondria ; r, dilated endoplasmic reticulum . X 65,000.
FIGURE 13
(arrows) . These areas resemble the dark bodies of myofilament bundles .
CAMPBELL, UEHARA, MARK, AND BURNSTOCK
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X 75,000.
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VOLUME 49, 1971
Published April 1, 1971
gap of 500-800 A between most adult cells (8) .
Regions of close apposition forming nexuses
were occasionally observed between muscle
cells (Fig . 17) . The outer leaflets of the membrane
were separated by a gap of up to 30 A, except
for a few short areas of fusion . These are, therefore, best described as "gap junctions" and have
also been shown to occur in adult smooth muscle
(38, 50) . The inner leaflets of the membranes
of these junctions were lined with electronopaque material .
Sorokin (47) described the process of ciliogenesis in both smooth muscle and fibroblasts .
Some evidence of this process was seen in cultured smooth muscle .
Membrane-bound particles about 1000 A in
diameter, containing an electron-opaque core,
were seen in some cultures . These particles resembled those seen in embryonic tissue (3) and
were quite similar to viruses associated with
Marek's disease (30) .
Cultured smooth muscle shows many of the characteristic features of in vivo embryonic smooth
muscle (3, 54), of smooth muscle in response to
estrogen (17, 43), and of regenerating smooth
muscle of anterior eye chamber transplants?
These features include an abundance of free
ribosomes, dilated rough endoplasmic reticulum,
and Golgi apparatus, all suggesting active protein
synthesis .
One must be cautious in applying results from
in vitro studies to in vivo studies . There may be
differences in fine structure due to the artificially
imposed culture milieu, and there may also be
s
Campbell, G. R ., Y . Uehara, T . Malmfors, and G .
Burnstock. 1970 . Degeneration and regeneration of
smooth muscle transplants in the anterior eye
chamber : an ultrastructural study . Z . Zellforsch .
Mikrosk . Anat . In Press.
Dense areas (da) with a well-defined substructure (circle) prominent along an infolding of the
cell membrane. Myofilaments seem to pass into the dense-area matrix . The extracellular space of the
membrane infoldings contains filamentous substance resembling basal lamina material (f) . What appear
to be thick (double headed arrow) and thin (arrow) myofilaments can be distinguished . X 80,000.
FIGURE 16
FIGURE 17 Gap junction between two cultured smooth muscle cells (S, , SI) . Inset shows high magnification of part of this junction . A gap of up to about 30 A can be seen between the outer leaflets of the unit
membrane ; there are a few short areas of fusion (arrows) . The inner leaflets of the membranes are lined
by an accumulation of electron-opaque material . X 480,000. Inset, X 560,000 .
CAMPBELL, UEHARA, MARK, AND BURNSTOCK
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DISCUSSION
differences related to factors such as lack of
innervation . However, cultured smooth muscle
has the particular advantage that it can be used
to study the relation of ultrastructural features
to functional observations on the same cell .
A number of authors have suggested that polysomes or free ribosomes play a major role in the
synthesis of myofilaments in striated muscle
(4, 7, 18, 19, 52, 53) . Isolated polysomes, containing 60-70 ribosomes, from 14 day old embryonic chick skeletal muscle, have been shown
to synthesize myosin (20, 21) . By analogy, the
large aggregates of ribosomes seen in both embryonic and cultured smooth muscle may also
be involved in myosin synthesis . Leeson and Leeson (28) describe the occurrence of polysomes and
free ribosomes in developing smooth muscle .
These become less marked in later development .
Yamauchi and Burnstock (54) noted a similar
transient appearance of ribosomal rosettes and
correlated this with the first appearance of
coarse myofilaments (60 A in diameter) and
dark bodies, although fine filaments (less than
30 A in diameter) were established earlier . In
these studies no definite evidence of structural
connections between polysomes and myofilaments
was shown .
Although myosin is present in smooth muscle
(31) and follows a pattern of aggregation similar
to that of myosin of striated muscle (24), the
question of the in vivo form of this myosin is a
subject of current debate . Kelly and Rice (25)
have shown that thick filaments are present in
contracted muscle and have suggested that these
are myosin filaments which aggregate during
contraction and disperse when the muscle is
relaxed . Panner and Honig (34, 35) were unable
to find these thick filaments in any stage of
contraction . They have, however, shown structures morphologically similar to myosin molecules and/or dimers in both the contracted and
Published April 1, 1971
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relaxed state . Only a few cultured smooth muscle formed at the cell membrane . Consequently,
cells contain thick myofilaments, the diameters another possibility is that dark bodies in smooth
of which are quite variable . This suggests : (a) muscle form from dense areas along the memthat thick filaments are either aggregations or brane or even from electron-opaque material
random superimpositions of thin filaments, (b) seen in association with some membranous
that cells within the same culture can be fixed systems .
Elaborate networks of tubules, similar to those
in different stages of contraction, (c) that
cells within the same culture can be at different found in cultured smooth muscle, have also
stages of differentiation, as Bennett and Cobb been observed in denervated rat skeletal muscle
(3) saw only thin myofilaments in the early (37), in plastids of some plants (32), and in the
stages of development of chicken gizzard .
chloride cells of the fish gill (14) . The functional
Randomly orientated networks of filaments significance of these networks reamains to be
(100 A filaments) are a feature of cultured smooth understood . However, Ishikawa (22) suggests
muscle . Similar filament arrangements have that similar networks of tubules found in both
also been seen in smooth muscle of 10 day old cultured and atrophied skeletal muscle are due
chick embryo gizzard, and these filaments are to abnormal T-system proliferation, and that
also present in adult tissue but in far smaller these may be indirectly due to the absence of
innervation . The same could be true of cultured
numbers .' Cytoplasmic filaments have also been
observed in a variety of other tissues . Salazar smooth muscle . Further studies need to be made
and Totten (44) noted patches of filaments in to understand the true function of these networks .
Myofilaments have often been reported to be
cells of human gastric leiomyoblastomas . However, these were 60-80 A in diameter and were attached to dense areas on the cell membrane of
thought to be actin . Similar filaments have also adult smooth muscle (26, 34, 36, 39, 40, 42) .
been described in rat liver epithelial cells (5), These dense areas presumably act as points of
in cells of blood-forming organs (48), and in attachment upon which myofilaments can demononuclear phagocytes (12), where their func- velop tension . In cultured smooth muscle, dense
tion may be cytoskeletal . Ishikawa et al . (23), areas along infoldings of the cell membrane probalthough unable to determine the exact nature ably play a similar role, since myofilament bunand function of the 100 A filaments in cultured dles seldom extend to the cell margins but are
skeletal muscle, provided evidence that they confined to patches within the cell .
were not involved in myofibrillogenesis . The
Nexuses appear to be the morphological basis
same may apply to smooth muscle . They did, of electrical interaction between individual smooth
however, find evidence which supported the muscle cells in adult organs (8) . However, they
theory of O'Brien and Thimann (33), who may have other or additional functions, since
suggest that microtubules and filaments are they have been described between fibroblasts in
alternate states of assembly of the same subunit tissue culture (13), and between protrusions of the
particle . In cultured smooth muscle evidence to same smooth muscle cell in guinea pig ureter .4 An
support this theory was not found . Nevertheless, interesting feature with respect to the nexuses
a close relationship between 100 A filaments found in cultured smooth muscle is that most of
and structures very similar in appearance to the cells were separated by enzyme dissociation
in the culturing procedure, so that any nexuses
dark bodies was evident . If these structures are
dark bodies, this suggests that the filaments present were likely to be newly formed .
play a role in dark body formation and/or form
. Merrillees
part of their substructure . A number of authors The authors are most grateful to N. C . R
and T. Bennett for their constructive criticism of the
have suggested that dark bodies correspond in
manuscript, and to Janet McConnell and to Vicki
function to the Z lines of skeletal muscle (8) . James for their excellent technical assistance .
The possibility therefore arises that they may be
This study was supported by grants from the
derived in a similar manner . Heuson-Stiennon Australian Research Grants Committee and the
(19) suggests that in embryonic rat skeletal musNational Heart Foundation of Australia .
cle Z lines appear to form from dense bodies Received for publication 6 July 1970, and in revised form
13 October 1970 .
' Uehara, Y., G . R. Campbell, and G . Burnstock.
1971 . Cytoplasmic filaments in developing and adult 4 Uehara, Y., and G. Burnstock . 1970 . Fine structure
vertebrate smooth muscle . Accepted J. Cell . Biol . of guinea-pig ureter . In preparation.
Published April 1, 1971
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